Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It Is estimated that over 150 million people worldwide are Infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so-called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system. In addition, the roles of cellular and humoral immune responses in protection against HCV infection and disease have yet to be established. Immunoglobulins have been reported for prophylaxis of transfusion-associated viral hepatitis, however, the Center for Disease Control does not presently recommend. Immunoglobulins treatment for this purpose. The lack of an effective protective immune response is hampering the development of a vaccine or adequate post-exposure prophylaxis measures, so in the near-term, hopes are firmly pinned on antiviral interventions. Various clinical studies have been conducted with the goal of identifying pharmaceutical agents capable of effectively treating HCV infection in patients afflicted with chronic hepatitis C. These studies have involved the use of interferon-alpha, alone and in combination with other antiviral agents. Such studies have shown that a substantial number of the participants do not respond to these therapies, and of those that do respond favorably, a large proportion were found to relapse after termination of treatment.
Until recently, Interferon (IFN) was the only available therapy of proven benefit approved in the clinic for patients with chronic hepatitis C. However the sustained response rate is low, and interferon treatment also induces severe side-effects (i.e. retinopathy, thyroiditis, acute pancreatibs, depression) that diminish the quality of life of treated patients. Recently, interferon in combination with ribavirin has been approved for patients non-responsive to IFN alone. However, the side effects caused by IFN are not alleviated with this combination therapy. Therefore, a need exists for the development of effective antiviral agents for treatment of HCV infection that overcomes the limitations of existing pharmaceutical therapies.
HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The single strand HCV RNA genoride is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. In the case of HCV, the generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one, as yet poorly characterized, cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (henceforth referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation of the NS3 protein with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV. A general strategy for the development of antiviral agents is to inactivate virally encoded enzymes that are essential for the replication of the virus. In this vein, patent application WO 97/06804 describes the (-) enantiomer of the nucleoside analogue cytosine-1,3-oxathiolane (also known as 3TC) as active against HCV. This compound, although reported as safe in previous clinical trials against HIV and HBV, has yet to be clinically proven active against HCV and its mechanism of action against the virus has yet to be reported. Intense efforts to discover compounds which inhibit the NS3 protease or RNA helicase of HCV have led to the following disclosures:
U.S. Pat. No. 5,633,388 describes heterocyclic-substituted carboxamides and analogues as being active against HCV. These compounds are directed against the helicase activity of the NS3 protein of the virus but clinical tests have not yet been reported. PA1 A phenanthrenequinone has been reported by Chu et al., (Tet. Lett., (1996), 7229-7232) to have activity against the HCV NS3 protease in vitro. No further development on this compound has been reported. PA1 A paper presented at the Ninth International Conference on Antiviral Research, Urabandal, Fukyshima, Japan (1996) (Antiviral Research, (1996), 30, 1, A23. (abstract 19)) reports thiazolidine derivatives to be inhibitory to the HCV protease. PA1 WO 98/17679 from Vertex Pharmaceuticals Inc. discloses inhibitors of serine protease, particularly, Hepatitis C virus NS3 protease. These inhibitors are peptide analogues based on the NS5A/5B natural substrate. Although several tripeptides are disclosed, all of these peptide analogues contain C-terminal activated carbonyl function as an essential feature. These analogues were also reported to be active against other serine protease and are therefore not specific for HCV NS3 protease. PA1 Hoffman LaRoche has also reported hexapeptides that are proteinase inhibitors useful as antiviral agents for the treatment of HCV Infection. These peptides contain an aldehyde or a boronic acid at the C-terminus. PA1 Steinkuhler et al. and Ingallinella et al. have published on NS4A-4B product inhibition (Biochemistry (1998), 37, 8899-8905 and 8906-8914). However, the peptides and peptide analogues presented do not include nor do they lead to the design of the peptides of the present invention. PA1 or B is an acyl derivative of formula R.sub.4 --C(O)--; a carboxyl derivative of formula R.sub.4 --O--C(O)--; an amide derivative of formula R.sub.4 --N(R.sub.5)--C(O)--; a thioamide derivative of formula R.sub.4 --N(R.sub.5)--C(S)--; or a sulfonyl derivative of formula R.sub.4 --SO.sub.2 wherein PA1 Y is H or C.sub.1-6 alkyl; PA1 R.sup.3 is C.sub.1-8 alkyl, C.sub.3-7 cycloalkyl, or C.sub.4-10 alkylcycloalkyl, all optionally substituted with hydroxy C.sub.1-6 alkoxy, C.sub.1-6 thioalkyl, amido, (lower alkyl)amido, C.sub.6 or C.sub.10 aryl, or C.sub.7-16 aralkyl; PA1 R.sub.2 is CH.sub.2 --R.sub.20, NH--R.sub.20, O--R.sub.20 or S--R.sub.20, wherein R.sub.20 is a saturated or unsaturated C.sub.3-7 cycloalkyl, or C.sub.4-10 (alkylcycloalkyl), all of which being optionally mono-, di- or tri-substituted with R.sub.21, or R.sub.20 is a C.sub.6 or C.sub.10 aryl or C.sub.7-14 aralkyl, all optionally mono-, di- or tri-substituted with R.sub.21, or R.sub.20 is Het or (lower alkyl)-Het, both optionally mono-, di- or tri-substituted with R.sub.21, PA1 R.sup.1 is H, C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl, all optionally substituted with halogen; or a pharmaceutically acceptable salt or ester thereof. PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, hydroxy or C.sub.1-6 alkoxy, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, amido, (lower alkyl)amide, or- amino optionally substituted with C.sub.1-6 alkyl; PA1 (v) Het or (lower alkyl)-Het, both optionally substituted with C.sub.1-6 alkyl, hydroxy, amino optionally substituted with C.sub.1-6 alkyl, amido, (lower alkyl)amide, or amino optionally substituted with C.sub.1-6 alkyl. PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy, amino optionally mono- or di-substituted with C.sub.1-6 alkyl, amido or (lower alkyl)amide; PA1 (ii) C.sub.3-7 cycloalkyl, C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amino optionally mono- or di-substituted with C.sub.1-6 alkyl, amido or (lower alkyl)amide; PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl optionally substituted with C.sub.1-6 alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; or PA1 (v) Het or (lower alkyl)-Het, both optionally substituted with C.sub.1-6 alkyl, hydroxy, amino optionally mono- or di-substituted with C.sub.1-6 alkyl, amido or (lower alkyl)amido. PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy, amido, (lower alkyl)amido, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amido, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (iii) amino optionally mono- or di-substituted with C.sub.1-3 alkyl; PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionally substituted with C.sub.1-6 alkyl; or PA1 (v) Het or (lower alkyl)-Het, both optionally substituted with C.sub.1-6 alkyl, hydroxy, amino optionally substituted with C.sub.1-6 alkyl, amido or (lower alkyl)amide; and PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl or C.sub.1-6 alkoxy; PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amino or amido. PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, hydroxy or C.sub.1-6 alkoxy; or PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl, (C.sub.1-6 alkoxy)carbonyl, such that B is, for example: ##STR16## PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, such that B is for example: ##STR17## PA1 (v) Het optionally substituted with C.sub.1-6 alkyl, hydroxy, amido or amino, such that B is for example: ##STR18## PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy or amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl, C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl, such that B is for example: ##STR19## PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, amino optionally substituted with C.sub.1-6 alkyl; or PA1 (v) Het or (lower alkyl)-Het, both optionally substituted with C.sub.1-6 alkyl, hydroxy, amido, or amino optionally mono-substituted with C.sub.1-6 alkyl, such that B is for example: ##STR20## PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; and PA1 (iii) amino optionally mono- or di-substituted with C.sub.1-3 alkyl, such that B is for example: ##STR22## PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, amino or amido optionally substituted with C.sub.1-6 alkyl; or PA1 (v) Het optionally substituted with C.sub.1-6 alkyl, hydroxy, amino or amido, such that B is for example: ##STR23## PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; ##STR25## PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl optionally substituted with C.sub.1-6 alkyl, hydroxy, amino or amido, such that B is for example: ##STR26## PA1 C.sub.1-6 alkoxy such as methoxyl, ##STR33## PA1 lower thioalkyl schwas ##STR34## PA1 halo such as chloro; PA1 amino optionally monosubstituted with C.sub.1-6 alkyl; or C.sub.6 or C.sub.10 aryl, such that R.sub.21A is for example: dimethylamino, Ph--N(Me)--; PA1 unsubstituted C.sub.6 or C.sub.10 aryl, C.sub.7-16 aralkyl, such as for example phenyl or ##STR35## PA1 or R.sub.21A is more preferably Het optionally substituted with R.sub.22 wherein R.sub.22 is C.sub.1-6 alkyl, C.sub.1-6 alkoxy, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl, or Het, such that R.sub.21A is for example: ##STR36## PA1 B is a C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substituted with C.sub.1-6 alkyl; or PA1 Het or (lower alkyl)-Het, all optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substituted with C.sub.1-6 alkyl, or PA1 B is R.sub.4 --SO.sub.2 wherein R.sub.4 is preferably amido; (lower alkyl)amide; C.sub.6 or C.sub.10 aryl, C.sub.7-14 aralkyl or Het, all optionally substituted with C.sub.1-6 alkyl, or PA1 B is an acyl derivative of formula R.sub.4 --C(O)-- wherein R.sub.4 is PA1 B is a carboxyl of formula R.sub.4 --O--C(O)--, wherein R.sub.4 is PA1 B is an amide of formula R.sub.4 --N(R.sub.5)--C(O)-- wherein R.sub.4 is PA1 B is thioamide of formula R.sub.4 --NH--C(S)--; wherein R.sub.4 is PA1 Y is H or methyl; PA1 R.sup.3 is C.sub.1-8 alkyl, C.sub.3-7 cycloalkyl, or C.sub.4-10 alkylcycloalkyl, all optionally substituted with hydroxy; C.sub.1-6 alkoxy, C.sub.1-6 thioalkyl, acetamido, C.sub.6 or C.sub.10 aryl, or C.sub.7-16 aralkyl; PA1 R.sub.2 is S--R.sub.20 or O--R.sub.20 wherein R.sub.20 is preferably a C.sub.6 or C.sup.10 aryl, C.sub.7-16 aralkyl, Het or --CH.sub.2 --Het, all optionally mono-, di- or tri-substituted with R.sub.21, wherein PA1 R.sup.2 is selected from the group consisting of: ##STR44## PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, hydroxy or C.sub.1-6 alkoxy; or PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl, (C.sub.1-6 alkoxy)carbonyl; or PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy; or PA1 (v) Het optionally substituted with C.sub.1-6 alkyl, hydroxy, amido or amino; PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy or amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl, C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; or PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, amino optionally substituted with C.sub.1-6 alkyl; or PA1 (v) Het or (lower alkyl)-Het, both optionally substituted with C.sub.1-6 alkyl, hydroxy, amido, or amino optionally mono-substituted with C.sub.1-6 alkyl; PA1 or B is an amide of formula R.sub.4 --N(R.sub.5)--C(O)-- wherein R.sub.4 is PA1 B is a thioamide of formula R.sub.4 --NH--C(S)--; wherein R.sub.4 is: PA1 B is an amide of formula R.sub.4 --NH--C(O)-- wherein R.sub.4 is PA1 Y is H.; PA1 R.sup.3 is the side chain of tert-butylglycine (Tbg), lle, Val, Chg or: ##STR46## PA1 R.sup.2 is 1-naphtylmethoxy; or quinolinoxy unsubstituted, mono- or di-substituted with PA1 R.sub.21 as defined above, or PA1 R.sup.2 is; ##STR47## PA1 P1 is a cyclopropyl ring wherein carbon 1 has the R configuration, ##STR48## PA1 B is tert-butoxycarbonyl (Boc) or ##STR49## PA1 R.sup.3 is the side chain of Tbg, Chg or Val; PA1 R.sup.2 is; ##STR50## PA1 2-phenyl-4-hydroxyquinoline derivatives IIIc were prepared according to Giardina et al. (J. Med. Chem., (1997), 40, 1794-1807). ##STR59## PA1 1) When R.sub.20 is aryl, aralkyl, Het or (lower alkyl)-Het, the process can be carried out according to the procedure described by E. M. Smith et al. (J. Med. Chem. (1988), 31, 875-885). Briefly, commercially available Boc-4(R)-hydroxyproline is treated with a base such as sodium hydride or potassium tert-butoxide and the resulting alkoxide reacted with halo-R.sup.20 (Br--R.sup.20, I--R.sub.20, etc.) to give the desired compounds. Specific embodiments of this process are presented in Examples 4, 5 and 7. PA1 2) Alternatively, when R.sub.20 is aryl or Het, the compounds can also be prepared via a Mitsunobu reaction (Mitsunobu (1981), Synthesis, January, 1-28; Rano et al., (1995), Tet. Lett. 36(22), 3779-3792; Krchnak et al., (1995), Tet. Lett. 36(5). 62193-6196; Richter et al., (1994), Tet. Lett. 35(27), 4705-4706). Briefly, commercially available Boc-4(S)-hydroxyproline methyl ester is treated with the appropriate aryl alcohol or thiol in the presence of triphenylphosphine and diethylazodicarboxylate (DEAD) and the resulting ester is hydrolyzed to the acid. Specific embodiments of this process are presented in Examples 6 and 8. ##STR63## PA1 1) enzymatic separation (Examples 13, 17 and 20); PA1 2) crystallization with a chiral acid (Example 18); or PA1 3) chemical derivatization (Example 10). PA1 coupling a peptide selected from the group consisting of: APG-P3-P2; or APG-P2; PA1 with a P1 intermediate of formula: ##STR68## PA1 wherein R.sup.1 is C.sub.1-6 alkyl, cycloalkyl or C.sub.2-6 alkenyl, all optionally substituted with halogen, CPG is a carboxyl protecting group and APG is an amino protecting group and P3 and P2 are as defined above. PA1 coupling a (suitably protected) amino acid, peptide or peptide fragment with a P1 intermediate of formula: ##STR69## PA1 wherein R.sup.1 is C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl or C.sub.2-6 alkenyl, all optionally substituted with halogen, and CPG is a carboxyl protecting group. PA1 coupling a (suitably protected) amino acid, peptide or peptide fragment with an intermediate of formula: ##STR70## PA1 wherein CPG is a carboxyl protecting group.
Several studies have reported compounds inhibitory to other serine proteases, such as human leukocyte elastase. One family of these compounds is reported in WO 95/33764 (Hoechst Marion Roussel, 1995). The peptides disclosed in this application are morpholinylcarbonyl-benzoyl-peptide analogues that are structurally different from the peptides of the present invention.
One advantage of the present invention is that it provides tripeptides that are inhibitory to the NS3 protease of the hepatitis C virus.
A further advantage of one aspect of the present invention resides in the fact that these peptides specifically inhibit the NS3 protease and do not show significant inhibitory activity at concentrations up to 300 .mu.M against other serine proteases such as human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), or bovine pancreatic chymotrypsin, or cysteine proteases such as human liver cathepsin B (Cat B).
A further advantage of the present invention is that it provides small peptides of low molecular weight that may be capable of penetrating cell membranes and may be active in cell culture and in vivo with good pharmacokinetic profile.